Methods and apparatus are disclosed for determining the volume, hemoglobin concentration, maturity and cell shape of mammalian red blood cells in a sample and simultaneously monitoring system standardization. Methods for distinguishing red blood cells from other cellular particles, prior to the red blood cell analysis are also disclosed. The method can be applied with accuracy over a wide range of visible spectrum. A whole blood sample is treated with a reagent solution containing a nonionic surfactant in an isotonic buffered solution at neutral pH, the red blood cells are passed through a beam of light in single file at a selected wavelength, obtaining an initial cytogram by means of the resultant magnitude of one light loss signal and one forward angle light scatter signal at a selected angular interval and a third side angle light scatter or two forward angle light scatter signals at selected angular intervals and a third side-angle light scatter signal, projecting the cytogram, point by point, onto a pre-calibrated 3-dimensional surface containing grid lines of volume and hemoglobin concentration, determining accurate values of cell volume and hemoglobin concentration by means of the location of each projected intercept onto the three dimensional grid surface.
1. Field of the Invention
The present invention relates to a method and apparatus for simultaneous monitoring of system standardization and automated analysis of mammalian red blood cell (RBC) and white blood cell (WBC) differentiation in a body fluid. The present invention particularly relates to a multi-angle light scatter and fluorescence apparatus such as multi-parameter hematology analyzer or flow cytometer that can perform both RBC and WBC differential analysis using the same optical detection system. The present invention more particularly relates to (i) a method for RBC analysis for volume, hemoglobin content, cell shape, and maturity in whole blood; (ii) an accurate method for determination of immature RBC (reticulocyte) volume and hemoglobin content; (iii) a RBC method that can continuously monitor the system standardization while a blood sample is being analyzed for RBC differentiation; and (iv) a method that can measure both mature RBC and reticulocyte volume and hemoglobin content, using one reagent and (v) an apparatus that can perform both WBC and RBC differential analysis using the same optical detection system.
2. Description of Prior Art
The conventional hematology method, microscopic examination of patient blood smears for RBC morphology for cell size, cell shape, color (for hemoglobin content) and inclusions provides a wealth of information leading towards the diagnosis and monitoring patient""s clinical conditions. Quite misleading impressions can be drawn, however, from substandard blood films, besides the fact that this manual method is very subjective and time consuming. During the past three decades, a number of automated hematology analyzers have become available to handle heavy laboratory work loads and to reduce labor. Most of these instruments measure mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) of red blood cells either by electrical impedance measurement or by light scatter optical measurement in combination with a colorimetric hemoglobin measurement. Because of incompleteness or ambiguity of morphological information in cell analysis from these systems, 5 to 10 percent of samples in hematology laboratories routinely undergo smear review for cell morphology using the microscopic method. More advanced hematology analyzers in terms of RBC morphology analysis are the Technicon H*1 and the Bayer ADVIA. Both systems are designed to measure red cell volume and hemoglobin concentration simultaneously on cell-by-cell basis, according to the teachings of D. H. Tycko, described in U.S. Pat. No. 4,735,504.
U.S. Pat. No. 4.735,504 to D. H. Tycko describes Method and Apparatus for Determining the Volume and Index of Refraction of Particles. He discloses the method for measuring V and HC of is ovolumetrically-sphered RBCs by 2 selected angular interval forward light scattering signals, S1 and S2, to determine volume (V) and hemoglobin concentration (HC). Drawbacks of the method are: 1) the wavelength of the light source must be long enough (e.g., 633 nm) to avoid hemoglobin absorption from RBCs, which precludes the choice of a light source more suitable for multi-parameter blood cell analysis (e.g., a 488 nm light source); 2) the two-dimensional (2D) matrix does not provide any information on abnormal cell shape since the signals from such cells fall on a wrong location on the predetermined 2D matrix, thus generating incorrect clinical data on V and HC; 3) the 2D matrix does not provide any information regarding shifts in the system standardization, the phenomenon that can occur without any warning due to an instability in fluidics passage caused by clots in certain blood samples or instability in electronics of the system; 4) the 2D scatter method is not capable of identifying and clearly separating WBC""s and nucleated red blood cells (NRBCs) from mature RBC""s or stained reticulocytes. WBC""s and NRBC""s generate much more scatter than RBC""s because of their nuclei and if they are not excluded cleanly from the RBC population before V and HC analysis, clinical results on MCV, hematocrit (Hct), MCHC and mean corpuscular hemoglobin (MCH) will be very misleading on elevated WBC or NRBC samples.
U.S. Pat. No. 5,194,909 to D. H. Tycko teaches Apparatus and Method for measuring V and HC of Red Blood Cells. The difference of this art from that of his previous teachings in U.S. Pat. No. 4,735,504 is that the 2D matrix is created using one forward light scattering signal (pre-selected) at a long wavelength (633 nm) and the second signal from a resistant pulse-sizing aperture. Drawbacks of the method are: 1) the method requires two independent sources of detection system, which creates unnecessary complications such as synchronization of the two signals from two different detection systems; 2) the wavelength of the light source must be long enough to avoid hemoglobin absorption from RBCs, which limits the choice of light source for multi-parameter blood cell analysis; 3) the 2D matrix does not provide any information on abnormally shaped RBCs, thus generating incorrect clinical information on V and HC; 4) the 2D matrix does not provide any information regarding shifts in the system standardization, the phenomenon that can occur without any warning due to instability in electronics or fluidics as explained above.
U.S. Pat. No. 5,284,771 to Fan et al. discloses Reagent Compositions and their use in sphering cells. The reagent composition includes a zwitterionic surfactant for sphering red blood cells to eliminate orientation noise and Ozxazine750 to stain reticulocytes. The light source of the optical detection system is a 633 nm HeNe laser, and the stained reticulocytes are identified by light scatter/absorption technology. Fluorescent measurement of retoculocytes was not demonstrated or claimed in this patent. The inventors of this disclosure did not make any claims on reticulocyte V and HC measurements, but they described the use of the aforenoted methods of Tycko to simultaneously measure the red cell volume and hemoglobin on a cell-by-cell basis using the TECHNICON H*1 SYSTEM. In the teachings of Fan et al., the reticulocyte staining procedure requires manual preparation, manual feeding, and over 2 min. of staining time. The inventors described that V and HC of both RBCs and reticulocytes are measured by the method of Tycko, although the reagents used for RBCs and reticulocytes are completely different in composition. The reagent used to construct Tycko""s 2D matrix for RBCs for the TECHNICON H*1 spheres and fixes the RBCs as described in Tycko""s disclosure, while the reagent used for the reticulocytes spheres RBCs in a buffer that does not contain any fixative. Besides, absorption by the blue dye used to stain reticulocytes interferes with the magnitude of the scatter signals in the measurement of V and HC of the stained cells. Aforementioned problems may lead to erroneous clinical data on reticulocyte V and HC measurements by the teachings of Fan et al. As mentioned above, Tycko""s method for V and HC measurement requires a light source which emits monochromatic light in a region where hemoglobin is very transparent. This limits the availability of the light source (xcexmax must be  greater than 600 nm, such as a red HeNe laser). Another limitation is the choice of available dyes, since the same wavelength must be used for the absorption, or to excite the dye for fluorescent emission. Under this condition, the dye must be a blue dye with a strong absorption of red light. No claims were made on the Reticulocyte VandHC measurement in this patent.
U.S. Pat. No. 5,350,695 to Collela et al. discloses the same methods and reagents for characterizing reticulocytes as previously disclosed in U.S. Pat. No. 5,284,771, except that a method of adjusting the measured absorption signals for pseudo-absorption is added. According to the description in the first paragraph, column 12, of this disclosure, the major problem of the invention disclosed in U.S. Pat. No. 5,284,771 is the absorption signals of TECHNICON H*1 instrument being at the same level as the noise of the absorption preamplifier. Therefore, they had to develop a mathematical model to improve the signal-to-noise (S/N) ratio of the absorption signals from the stained reticulocytes. Even with the mathematical correction disclosed in this patent, generating satisfactory SIN ratio of absorption signals from the stained reticulocytes appears to be the major problem of this method. Besides, light absorption will also interfere with the magnitude of light scatter of the particle.
U.S. Pat. No. 5.360,739 to Fan et al. discloses the methods and reagents as disclosed in the four previous patents, except that the blue excitable fluorescent dye, acridine orange, is included. To practice the teachings of this method, it requires two light sources, one for the blue excitable fluorescent measurement (Argon/Ion laser) and another for cell volume and hemoglobin measurement (HeNe laser). A drawback of this invention is making the detection system unnecessarily complicated for synchronization and standardization and increases the instrument production cost significantly.
U.S. Pat. No. 5,438,003 to Collela et al. discloses the same reagent compositions disclosed in the 4 previous patents for use in the identification and characterization of reticulocytes in whole blood. All the claims of this invention are related to reagent composition and no claims are made on the method of V and HC measurement of reticulocytes. However, they have a new and lengthy explanation in the text how they are correcting xe2x80x9cpseudo-absorptionxe2x80x9d and hemoglobin interference in the disclosed method in separating reticulocytes from mature RBCs by light scatter/absorption utilizing a HeNe (633 nm) light source. Consequently, the cytograms presented in U.S. Pat. No. 5,43 8,003 reveal reticulocyte signals poorly separated from that of mature RBCs. It will be very difficult to accurately measure V and HC of reticulocytes unless the population is well separated from mature RBCs by the disclosed light scatter/absorption method.
Furthermore, the examples in this disclosure reveal that the disclosed method requires two reagents which are completely different from the reagent used to construct the 2D map for VandHC measurements, in pH, osmolarity, sphering agent, and buffer. In addition, the H*1 RBC method has a very short incubation time, whereas the staining time for reticulocytes in the disclosed reagent is 2 min. (30 seconds vs. 120 seconds). The RBC sphering process is a very sensitive and reversible process, requiring precise timing to obtain consistently reproducible V and HC data based on the two selected light scatter signals. Collela et al. neither disclose any time study data on V and HC nor disclose any reticulocyte V and HC data in this patent.
Given these aspects of prior art, it is desirable to offer an improved method and apparatus for complete RBC differential analysis and more accurate reticulocyte count and V and HC measurement. It is an object of the present invention to provide an apparatus that can perform both WBC and RBC differential analysis using a single light source and optical detection system. It is another object of the present invention to provide more complete RBC differential analysis that includes clinically useful MCHC measurement on cell-by-cell bases and detection and quantitation of abnormal shape RBCs. It is yet another object of the present invention is to provide a single reticulocyte reagent that can be used for VandHC measurement of both mature RBCs and reticulocytes. Further object of the present invention is to provide a method that does not limit the choice of the light source within the very narrow region. Yet another object of the present invention is to provide an RBC/diff method for continuous monitoring of the system stability. These and further objects of the invention will become apparent to those of ordinary skill in the art from the following descriptions and figures.
The present invention relates to (1) a method and apparatus for simultaneous monitoring of system standardization and automated analysis of RBC differentiation on a multi-angle light scatter and fluorescent hematology analyzer or flow cytometer; (2) an apparatus that can perform both WBC and RBC differential analyses using the same light source and the same optical detection system; (3) more particularly relates to a method for RBC analysis for volume, hemoglobin content, cell shape and maturity in whole blood or body fluid; (4) an accurate method for determination of reticulocyte (immature RBC) VandHC, useful for diagnosis of iron deficiency anemia in children and hemodialysis patients; (5) an RBC method that can continuously monitor the system standardization while a blood sample is being analyzed, the feature eliminates the possibility of reporting the wrong clinical data, caused by system drifts. The above goals are achieved using a well-defined and pre-calibrated 3-dimensional (3D) surface as the built-in measuring device, which is created using both theoretical and actual data (events) generated by the multi-angle light scatter and/or light loss measurements. Each event of normal RBCs should therefore fall upon this infinitesimally thin 3D surface. If a majority of cell signatures fall below or above the surface, creating another layer, it is an indication that the subject channel is out of standardization, either due to electronic or fluidic shift. A multi-dimensional analysis of three or more light scatter and/or light loss signals provides a degree of internal consistency. Abnormally shaped RBCs are defined by the closest distance of each event from the surface, since all normal RBC signals (sphered in the CELL DYN 4000 Diluent-Sheath) fall in close proximity to the surface. Reticulocytes are defined by the fluorescent intensity of each event treated with a nucleic acid stain in the CELL DYN 4000 Reticulocyte Reagent. The cell VandHC are determined based on the location of each event on the 3D matrix created by ALL/IASxe2x80x2/PSS or IAS/IASxe2x80x2/PSS. To achieve the above goals, the CELL DYN 4000 detector was modified to create 3 zones; the central rectangular zone (Zone 1) for axial light loss (0 degree, ALL) and the current CELL DYN 4000 intermediate angle (Zone 2), which is 2.24xc2x0-7.45xc2x0 (IAS), is divided into three sub-zones, where the central zone (Zone 3) is 4.5xc2x0-5.5xc2x0 (IASxe2x80x2). The modified detector will be referred to as a 3-Ring detector in this disclosure. The 3D surface is constructed using the 2 angles (ALL and IASxe2x80x2, or IAS and IASxe2x80x2) generated by the aforementioned 3-ring detector, and polarized 90xc2x0 side scatter (PSS) signal. The 3D principle is applicable to optical detection systems with a broad range of light source wavelengths (e.g., 488 nm, 532 nm or 633 nm).